Although the kidney is a complex organ with an intricate vascular supply and at least 15 different cell types, the critical functions of filtration, reabsorption and excretion can be targeted with tissue engineering. The basic functional unit of the kidney, the nephron, is composed of a vascular filter, the glomerulus, and a resorptive unit, the tubule. Filtration is dependent on flow and specialized glomerular endothelial cells. The majority (50-65%) of reabsorption is performed by the proximal tubule cells using active sodium transport through the energy-dependent Na+-K+-ATPase located on the basolateral membrane. Only 5-10% of the approximately one million nephrons in each human kidney is required to sustain normal excretory function.
The design of a tissue engineered renal replacement device can then be focused on the development of a glomerular endothelial filter in conjunction with a proximal tubule device for reabsorption and excretion. The endothelial filter is specifically designed to provide physiologic flow with low thrombogenicity and maximized surface area for solute transport. The proximal tubule device, containing an appropriate number of cells for renal replacement, has optimized surface area for solute reabsorption and an outlet for urine excretion. (See FIG. 27.) Several layers of molds and/or polymer scaffolds and semi-permeable membranes can be stacked to optimize filtration and reabsorption (FIG. 28). Biocompatible, bioresorbable and microporous polymers are used throughout for optimal cell growth and function.
Materials and Methods
Configuring the Mold
MEMS replica molding was used to create the polymer molds used in this Example (FIG. 2). Using the techniques described herein, an inverse pattern (i.e., protrusions rather than indentations) corresponding to the desired pattern of microchannels was formed on a silicon wafer. Poly-(dimethyl siloxane) (PDMS) was then cast onto the silicon template. After the template was removed, the PDMS was subjected to O2 plasma treatment, and was fastened to a second layer of PDMS. In this Example, the second layer of PDMS was flat, however, in other embodiments, either or both surfaces of the second PDMS layer can contain a pattern of microchannels. In addition, a semi-permeable membrane can be fastened between the PDMS layers.
Cell Culture
Renal proximal tubule cells and glomerular endothelial cells from rat and pig models have been isolated using sieve filtration and separation over a Percoll gradient (Vinay, et al. Am J Physiol 241, F403 (1981); Misra, et al. Am J Clin Path 58, 135 (1972)). Human microvascular endothelial cells were isolated from normal neonatal foreskin in collaboration with Dr. Michael Detmar (Cutaneous Biology Research Center, MGH Charletown), and stained positively for endothelial cell markers CD-31 and von Willebrand's factor (vWF) within the PDMS devices.
Both renal proximal tubule cells and human microvascular endothelial cells were seeded into the MEMS-designed PDMS (poly(dimethyl siloxane)) devices at 20 million cells/ml. Cells were allowed to adhere at 37° C. for six hours. Devices were rotated 180 degrees at three hours to allow adherence of cells to both sides of the microchannels. Flow was then started via infusion pump with appropriate culture medium to maintain cell viability.
Animal Model
The appropriate animal is made urernic via bilateral nephrectomies and connected to a hemoperfusion circuit. The tissue engineered renal replacement device is connected such that the venous blood enters the glomerular endothelial network and is returned to the animal. The fluid filtered through the glomerular network then passes through the proximal tubule network. Reabsorbed fluid is returned to the animal, while the fluid remaining in the proximal tubule lumen is analyzed as processed ultrafiltrate (urine). The goal rate of hemofiltration is 15-20 ml/min. to match the rate used in renal dialysis. Function of the renal replacement device is assessed as compared to matched controls for electrolytes, blood urea nitrogen and creatinine levels, glutathione reabsorption, ammonia excretion, and 1,25-(OH) 2 D3 levels.
Results
Human microvascular endothelial cells were seeded into poly (dimethyl siloxane) (PDMS) microchannels (smallest channel width 30 μm, depth 35 μm) using the specifically designed MEMS templates, and good cell adherence and proliferation within the channels was observed (FIG. 29).
A computational model is used to maximize blood flow through the glomerular cell filter, within normal hemodynamic parameters. Finite Element Modeling (FEM) technologies are used to maximize the surface area for filtration to simulate mass transport of solutes across the filter. The template topography and branching angles are designed to minimize thrombosis within the microchannels. Similarly, the proximal tubule network is optimized to provide even flow distribution, surface area for reabsorption, and an outflow tract for excretion of urine.
Cultured proximal tubular cells exhibit characteristic dome formation. Glomerular endothelial cells have also been isolated and maintained in culture. Further characterization of the cells is performed using immunohistochemical staining. Proximal tubule cells are stained for megalin (gp330) expression, and endothelial cells are stained for von Willebrand's factor (vWF) and CD-31.
Function of proximal tubule cells is assessed with the conversion of 1,25-OH-D3 to 1,25-(OH)2D3 (1,25-dihydroxyvitamin D3), the reclamation of glutathione and the generation of ammonium using a single pass perfusion system. 25-(OH) D3-12-hydroxylase is a cytochrome P-450 monooxygenase found in the inner mitochondrial membrane of proximal tubule cells. Proximal tubule glutathione reclamation is performed by the brush-border enzyme gamma-glutamyl transpeptidase. In addition, specific transport functions such as vectoral fluid transport (inhibited by ouabain, an Na+-K+-ATPase inhibitor), active bicarbonate and glucose transport (inhibited by acetazolamide and phlorizin respectively), and para-aminohippurate secretion (inhibited by probenecid) are also tested (Humes, et al., Kid Int 55, 2502 (1999); Humes, et al. Nat Biotechnol 17, 451 (1999)). Glomerular endothelial cell function is assessed for permeability to water and serum proteins, and the basement membrane components analyzed.
Microvascular endothelial and proximal tubule cells into have been successfully seeded into PDMS networks made from MEMS templates. FIGS. 30-32 show proximal tubule cells growing in the microchannels of the polymer scaffold at various intervals after seeding.
After generation of microporous biodegradable polymer templates, specific cell attachment and proliferation are tested using DNA or MTT assays. Cells on polymer templates are examined for confluence using histology and electron microscopy, as well as insulin leak rates (<10% infused). Enhanced attachment of proximal tubule and glomerular endothelial cells to polymers is optimized by precoating the polymer surface with several extracellular matrix components (Matrigel, collagen, fibronectin and laminin) or peptide sequences such as RGD, as described herein.
Flow studies are performed in glomerular endothelial and proximal tubule networks in vitro to simulate physiologic blood flow and hemodynamic parameters and to examine cell viability and function.